The development of novel approaches for transgene delivery into human airway epithelial cells has important implications towards gene therapy of Cystic Fibrosis lung disease. Although viral based vectors have paved the way for somatic gene transfer in CF and other diseased tissue, there are inherent concerns over their safety and immunogenicity. An alternative approach is to utilize an inert particle composed of a plasmid expression vector and a poly-L-lysine conjugate containing a specific receptor ligand. These DNA/protein complex structures are successfully endocytosed by cells that express the appropriate receptor, however, because of the normal vectorial movement of the receptosome to lysosomes the efficiency of gene transfer is low. One approach to circumvent lysosomal targeting is to add a endosomalytic agent to the DNA/protein complex thereby facilitating the release of lumenal contents into the cytoplasm. This proposal seeks to demonstrate the efficacy of enhancing DNA/protein complex transgene delivery through the action of an alpha-helical transmembrane domain from diphtheria toxin and exotoxin A. These two bacterial toxins are encoded as a single polypeptide and contain three domains that confer catalytic, endosome membrane spanning, and receptor- binding functions. The transmembrane domain is believed to mediate the translocation of the toxic peptide into the cytoplasm and it is likely that this results in membrane disruption. We propose that the degree to which this occurs is sufficient to cause "leakage" of the endosomal contents. In this study we will synthesize poly-L-lysine conjugates containing the transmembrane domain from diphtheria toxin and exotoxin A and determine their effectiveness in facilitating DNA/protein complex transgene delivery. A suitable receptor ligand for airway epithelial cells will also be identified and conjugated to poly-L-lysine. A ternary DNA/protein complex composed of the endosomalytic transmembrane and receptor ligand conjugates will be prepared and used to target epithelial CF cell lines. In vitro complementation of CFTR deficiency will be accomplished by including a CFTR expression vector in the ternary complex. The ability of the ternary complex to correctly target airway epithelium will be demonstrate in vivo using reconstituted human xenographs and a ferret animal model.